Nucleic acids and methods for the treatment of pompe disease

ABSTRACT

The invention relates to nucleic acids and methods for restoring acid alpha-glucosidase (GAA) activity in patients with Pompe disease using splice-switching technology.

FIELD OF THE INVENTION

The invention relates to nucleic acids and methods for restoring acidalpha-glucosidase (GAA) activity in patients with Pompe disease usingsplice-switching technology. In particular, the invention providesmethods to allow normal inclusion of exon 2 into the GAA mRNA in Pompepatients with the c.-32 IVS1-13 T>G mutation causing deleteriousskipping of exon 2, which encompasses the ATG start codon of the GAAtranscript. More specifically, the invention relates to a method forassembly of a functional GAA mRNA by means of antisense oligonucleotidescompatible with systemic delivery in order to access all affectedtissues throughout the body (skeletal muscles, heart and liver). Such anapproach meets the therapeutic needs of over half of all adult Caucasianpatients with Pompe disease.

BACKGROUND

Pompe disease (or glycogen storage disease type II—GSD II) is caused bypathogenic mutations in the acid α-glucosidase gene (GAA) located onchromosome 17. The GAA gene spans over 20,000 base pairs. It contains 20exons giving rise to a full length mRNA of about 3.6 kb, translated fromthe initiation codon located in exon 2 as an inactive precursor of about110 kD, which is further processed in mature forms of 70-77 kD. The modeof inheritance of the disease is recessive: patients have two pathogenicmutations in the acid α-glucosidase gene, one on each allele. Basically,the very nature of the mutations affecting the GAA gene together withthe combination of the mutant alleles determine the level of residuallysosomal acid α-glucosidase activity and subsequent clinical severity.In most cases, a combination of two alleles with fully deleteriousmutations leads to virtual absence of acid α-glucosidase activity and tothe severe classic infantile phenotype. A severe mutation in one alleleand a milder mutation in the other result in a slower progressivephenotype with residual activity up to 23% of average control activity.However, for these patients, enzyme activity is not always predictive ofthe age of onset and progression of the disease.

Hundreds of GAA mutations have been identified, but some are more commonamong given ethnic groups. For example:

-   c.-32 IVS1-13T>G is a splice mutation found in over half of all    adult Caucasian patients.-   Asp645Glu is found in most infants with Pompe disease from Taiwan.-   Arg854X nonsense mutation is found in many affected African or    African-American infants.-   del525T and del exon 18 are commonly seen in Dutch infants with the    disease.    Patients with the common “c.-32 IVS1-13T>G” mutation (a splicing    mutation lessening dramatically, but not completely, the inclusion    of exon 2 in the final transcript—leaky mutation), combined with a    fully deleterious mutation on the other allele, all show significant    residual enzyme activity and a protracted course of disease, but    onset of symptoms varied from the 1st year of life to late adulthood    (Kroos M, et al.; Am J Med Genet Part C Semin Med Genet 2012,    160C:59-68; Kroos M, et al., Neurology 2007, 68:110-115; Raben N, et    al.; Hum Mol Genet, 1996, 5(7):995-1000).

Pompe disease has long been an untreatable disorder, for which onlysupportive care was available. In March 2006, Myozyme (manufactured byGenzyme), the first treatment for patients with Pompe disease, receivedmarketing authorization in the European Union, followed in April 2006 byFDA approval in the United States. Myozyme is an “enzyme replacementtherapy” (ERT), which is supplied by intravenous delivery of the lackingenzyme. Another approach involves gene therapy. The rationale for genetherapy is to introduce a gene encoding the replacement enzyme into thesomatic cells, thus creating a permanent enzyme source. To this end, thecoding sequence for human acid α-glucosidase is inserted in a viralvector. For Pompe disease, gene therapy using adenoviral (Ad),Adeno-Associated (AAV) and hybrid Ad-AAV vectors have been investigatedin rat, mouse and quail. So far, preclinical results in animal modelsare encouraging, but sustained expression of the therapeutic transgene,prevention of antibody formation against the viral vector and/or acidα-glucosidase, as well as safety issues are still questionable. Anotherapproach used chaperone therapy. Some of the pathogenic mutations in theacid α-glucosidase gene lead to abnormal forms of the enzyme that arepoorly transported to the lysosome or are unstable in the lysosomalenvironment. The rationale for chaperone therapy is that some smallmolecules may have the property to stabilize and thus enhance theresidual acid α-glucosidase activity in the lysosomes of patients withthis type of mutations. The effect of chemical chaperones has so faronly been tested in cultured fibroblasts from patients with Pompedisease (Okumiya, Mol Genet Metab 2007; 90:49-57).

It is apparent that although different strategies have been proposed fortreating Pompe disease, as summarized above, there is still a need foran efficient therapeutic approach for treating the most common form ofthe disease.

SUMMARY OF THE INVENTION

The present invention relates to a nucleic acid 10 to 50 nucleotides inlength, complementary to a nucleotide sequence of the acidalpha-glucosidasc (GAA) pre-mRNA, said nucleic acid being able tocorrect the splicing of exon 2 of the acid alpha-glucosidase pre-mRNA.As will be shown below, the nucleic acid of the invention is anantisense oligonucleotide useful for treating Pompe disease. In aparticular embodiment, the nucleic acid comprises a nucleotide sequencecomplementary to an Exonic Silencer Sequence (ESS) present in exon 2 ofthe pre-mRNA encoding acid alpha-glucosidase.

In a particular embodiment, the nucleic acid molecule of the inventioncomprises or consists of a nucleotide sequence complementary to asequence comprised in the region defined by positions [+3;+45] of exon 2of the human GAA gene (SEQ ID NO:1), wherein preferably said sequencecomprised in the region defined by positions [+3;+45] of exon 2 of thehuman GAA gene (SEQ ID NO:1) has a length from 10 to 35 nucleotides,preferably from 13 to 35 nucleotides or from 10 to 30 nucleotides, andhas, for example, a length of 10, or 15, or 20 or 30 nucleotides, andwherein further preferably said nucleotides are contiguous nucleotidesin said region defined by positions [+3;+45] of exon 2 of the human GAAgene (SEQ ID NO:1).

In a further preferred embodiment, the nucleic acid molecule of theinvention comprises or consists of a nucleotide sequence complementaryto a sequence comprised in the region defined by positions [+3;+31] ofexon 2 of the human GAA gene (SEQ ID NO:1), wherein preferably saidsequence comprised in the region defined by positions [+3;+31] of exon 2of the human GAA gene (SEQ ID NO:1) has a length from 10 to 29nucleotides, preferably from 13 to 29 nucleotides, and has, for example,a length of 10, or 15, or 20 or 25 nucleotides, and wherein furtherpreferably said nucleotides are contiguous nucleotides in said regiondefined by positions [+3;+31] of exon 2 of the human GAA gene (SEQ IDNO:1).

In further preferred embodiment, the nucleic acid molecule of theinvention comprises or consists of a nucleotide sequence complementaryto a sequence comprised in the region defined by positions [+3;+31] ofexon 2 of the human GAA gene (SEQ ID NO:1), wherein preferably saidsequence comprised in the region defined by positions [+3;+31] of exon 2of the human GAA gene (SEQ ID NO:1) has a length from 10 to 29nucleotides, preferably from 13 to 29 nucleotides, and has, for example,a length of 10, or 15, or 20 or 25 nucleotides, and wherein saidnucleotides are contiguous nucleotides in said region defined bypositions [+3;+31] of exon 2 of the human GAA gene (SEQ ID NO:1).

Thus, in a preferred embodiment, the present invention provides for anucleic acid molecule, wherein said nucleic acid molecule is 10 to 50nucleotides in length and is complementary to a nucleotide sequence ofthe acid alpha-glucosidase (GAA) pre-mRNA, and wherein said nucleic acidmolecule is an antisense oligonucleotide and is able to correct thesplicing of exon 2 of the acid alpha-glucosidasc pre-mRNA, and whereinsaid nucleotide sequence of the GAA pre-mRNA is a sequence comprised inthe region defined by positions [+3;+45] of exon 2 of the human GAA gene(SEQ ID NO:1), and wherein preferably said sequence comprised in theregion defined by positions [+3;+45] of exon 2 of the human GAA gene(SEQ ID NO:1) has a length from 10 to 35 nucleotides, preferably from 13to 35 nucleotides or from 10 to 30 nucleotides, and has, for example, alength of 10, or 15, or 20 or 30 nucleotides, and most preferably saidsequence comprised in the region defined by positions [+3;+45] of exon 2of the human GAA gene (SEQ ID NO:1) has a length of 15 nucleotides.

In again further preferred embodiment, the present invention providesfor a nucleic acid molecule, wherein said nucleic acid molecule is 10 to50 nucleotides in length and is complementary to a nucleotide sequenceof the acid alpha-glucosidase (GAA) pre-mRNA, and wherein said nucleicacid molecule is an antisense oligonucleotide and is able to correct thesplicing of exon 2 of the acid alpha-glucosidase pre-mRNA, and whereinsaid nucleotide sequence of the GAA pre-mRNA is a sequence comprised inthe region defined by positions [+3;+45] of exon 2 of the human GAA gene(SEQ ID NO:1), wherein said antisense oligonucleotide is a tc-DNA or a2′-O-methyl-RNA, wherein the phosphate backbone of said antisenseoligonucleotide is composed of solely phosphodiester linkages, solelyphosphorothioate linkages or combinations of phosphodiester andphosphorothioate linkages. Preferably said antisense oligonucleotide isa tc-DNA or a 2′-O-methyl-RNA, wherein the phosphate backbone of saidantisense oligonucleotide is composed of solely phosphodiester linkages.Also preferred are embodiments, wherein said antisense oligonucleotideis a tc-DNA or a 2′-O-methyl-RNA, wherein the phosphate backbone of saidantisense oligonucleotide is composed of solely phosphorothioatelinkages. In another preferred embodiment, said antisenseoligonucleotide is a tc-DNA or a 2′-O-methyl-RNA, wherein the phosphatebackbone of said antisense oligonucleotide is composed of combinationsof phosphodiester and phosphorothioate linkages. Further preferably saidsequence comprised in the region defined by positions [+3;+45] of exon 2of the human GAA gene (SEQ ID NO:1) has a length from 10 to 35nucleotides, preferably from 13 to 35 nucleotides or from 10 to 30nucleotides, and has, for example, a length of 10, or 15, or 20 or 30nucleotides, and most preferably said sequence comprised in the regiondefined by positions [+3;+45] of exon 2 of the human GAA gene (SEQ IDNO:1) has a length of 15 nucleotides.

In again further preferred embodiment, the present invention providesfor a nucleic acid molecule, wherein said nucleic acid molecule is 10 to50 nucleotides in length and is complementary to a nucleotide sequenceof the acid alpha-glucosidase (GAA) pre-mRNA, and wherein said nucleicacid molecule is an antisense oligonucleotide and is able to correct thesplicing of exon 2 of the acid alpha-glucosidase pre-mRNA, and whereinsaid nucleotide sequence of the GAA pre-mRNA is a sequence comprised inthe region defined by positions [+3;+31] of exon 2 of the human GAA gene(SEQ ID NO:1), and wherein preferably said sequence comprised in theregion defined by positions [+3;+31] of exon 2 of the human GAA gene(SEQ ID NO:1) has a length from 10 to 29 nucleotides, preferably from 13to 29 nucleotides, and has, for example, a length of 10, or 15, or 20 or25 nucleotides.

In again further preferred embodiment, the present invention providesfor a nucleic acid molecule, wherein said nucleic acid molecule is 10 to50 nucleotides in length and is complementary to a nucleotide sequenceof the acid alpha-glucosidase (GAA) pre-mRNA, and wherein said nucleicacid molecule is an antisense oligonucleotide and is able to correct thesplicing of exon 2 of the acid alpha-glucosidase pre-mRNA, and whereinsaid nucleotide sequence of the GAA pre-mRNA is a sequence comprised inthe region defined by positions [+3;+31] of exon 2 of the human GAA gene(SEQ ID NO:1), wherein said antisense oligonucleotide is a tc-DNA or a2′-O-methyl-RNA, wherein the phosphate backbone of said antisenseoligonucleotide is composed of solely phosphodiester linkages, solelyphosphorothioate linkages or combinations of phosphodiester andphosphorothioate linkages. Preferably said antisense oligonucleotide isa tc-DNA or a 2′-O-methyl-RNA, wherein the phosphate backbone of saidantisense oligonucleotide is composed of solely phosphodiester linkages.Also preferred are embodiments, wherein said antisense oligonucleotideis a tc-DNA or a 2′-O-methyl-RNA, wherein the phosphate backbone of saidantisense oligonucleotide is composed of solely phosphorothioatelinkages. In another preferred embodiment, said antisenseoligonucleotide is a tc-DNA or a 2′-O-methyl-RNA, wherein the phosphatebackbone of said antisense oligonucleotide is composed of combinationsof phosphodiester and phosphorothioate linkages. Further preferably saidsequence comprised in the region defined by positions [+3;+31] of exon 2of the human GAA gene (SEQ ID NO:1) has a length from 10 to 29nucleotides, preferably from 13 to 29 nucleotides, and has, for example,a length of 10, or 15, or 20 or 25 nucleotides.

In particular, the nucleic acid of the invention may be complementary toone or more of the sequences defined by positions 3-17, 7-21 or 17-31 inSEQ ID NO: 1 (representing human GAA exon 2) or in a sequence having atleast 90% homology with SEQ ID NO:1. In a particular embodiment, thenucleic acid molecule of the invention comprises or consists of anucleotide sequence complementary to one or more of the sequencesdefined by positions [+3;+17], [+7;+21] or [+17;+31] in SEQ ID NO:1 orin a sequence having at least 90% homology with SEQ ID NO:1, inparticular at least 95%, more particularly at least 99%. In a furtherpreferred embodiment of the present invention, the nucleic acid moleculecomprises or consists of a nucleotide sequence complementary to one ormore of the sequences selected from (i) the sequence defined by position[+3;+17] of SEQ ID NO:1; (ii) the sequence defined by position [+7;+21]of SEQ ID NO:1; (iii) the sequence defined by position [+17;+31] of SEQID NO:1; (iv) a sequence having at least 90% homology, preferably atleast 95% homology, and further preferably at least 99% homology withthe sequence defined in either (i), (ii) or (iii). Further preferablysaid sequence has a length from 10 to 29 nucleotides, preferably from 13to 29 nucleotides, and has, for example, a length of 10, or 15, or 20 or25 nucleotides.

Thus, in a preferred embodiment, the present invention provides for anucleic acid molecule, wherein said nucleic acid molecule is 10 to 50nucleotides in length and is complementary to a nucleotide sequence ofthe acid alpha-glucosidase (GAA) pre-mRNA, and wherein said nucleic acidmolecule is an antisense oligonucleotide and is able to correct thesplicing of exon 2 of the acid alpha-glucosidase pre-mRNA, and whereinsaid nucleic acid molecule comprises or consists of a nucleotidesequence complementary to one or more of the sequences selected from (i)the sequence defined by position [+3;+17] of SEQ ID NO:1; (ii) thesequence defined by position [+7;+21] of SEQ ID NO:1; (iii) the sequencedefined by position [+17;+31] of SEQ ID NO:1; (iv) the sequence definedby position [+3;+31] of SEQ ID NO:1; (v) the sequence defined byposition [+3;+45] of SEQ ID NO:1; (vi) the sequence defined by position[+3;+46] of SEQ ID NO:1; (vii) a sequence having at least 90% homology,preferably at least 95% homology, and further preferably at least 99%homology with the sequence defined in any one of (i), (ii), (iii), (iv),(v) or (vi). In a further preferred embodiment, the present inventionprovides for a nucleic acid molecule, wherein said nucleic acid moleculeis 10 to 50 nucleotides in length and is complementary to a nucleotidesequence of the acid alpha-glucosidase (GAA) pre-mRNA, and wherein saidnucleic acid molecule is an antisense oligonucleotide and is able tocorrect the splicing of exon 2 of the acid alpha-glucosidase pre-mRNA,and wherein said nucleic acid molecule comprises or consists of anucleotide sequence complementary to one or more of the sequencesselected from (i) the sequence defined by position [+3;+17] of SEQ IDNO:1; (ii) the sequence defined by position [+7;+21] of SEQ ID NO:1;(iii) the sequence defined by position [+17;+31] of SEQ ID NO:1; (iv) asequence having at least 90% homology, preferably at least 95% homology,and further preferably at least 99% homology with the sequence definedin either (i), (ii) or (iii). In again a further preferred embodiment,the present invention provides for a nucleic acid molecule, wherein saidnucleic acid molecule is 10 to 50 nucleotides in length and iscomplementary to a nucleotide sequence of the acid alpha-glucosidase(GAA) pre-mRNA, and wherein said nucleic acid molecule is an antisenseoligonucleotide and is able to correct the splicing of exon 2 of theacid alpha-glucosidase pre-mRNA, and wherein said nucleic acid moleculecomprises or consists of a nucleotide sequence complementary to one ormore of the sequences selected from (i) the sequence defined by position[+3;+17] of SEQ ID NO:1; (ii) the sequence defined by position [+7;+21]of SEQ ID NO:1; or (iii) the sequence defined by position [+17;+31] ofSEQ ID NO:1.

In a particular embodiment, the nucleic acid of the invention comprisesor consists of the nucleotide sequence shown in SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4 or SEQ ID NO:5.

In a further preferred embodiment of the present invention, the nucleicacid molecule comprises or consists of a nucleotide sequencecomplementary to one or more of the sequences selected from (i) thesequence of SEQ ID NO:2; (ii) the sequence of SEQ ID NO:3; (iii) thesequence of SEQ ID NO:4; (iv) the sequence of SEQ ID NO:5; (v) thesequence of SEQ ID NO:12; or (vi) the sequence of SEQ ID NO:13.

In another aspect, the present invention provides for a nucleic acidmolecule being able to restore the normal splicing of exon 2 of thehuman acid alpha-glucosidase (GAA) pre-mRNA displaying the c.-32-13T>Gmutation, wherein said nucleic acid molecule comprises or consists of anantisense oligonucleotide, wherein said antisense oligonucleotide iscomplementary to a nucleotide sequence of said human acidalpha-glucosidase (GAA) pre-mRNA, wherein said nucleotide sequence ofsaid GAA pre-mRNA is a sequence comprised in the region defined bypositions [+3;+45] of exon 2 of the human GAA gene (SEQ ID NO:1), andwherein preferably said sequence comprised in the region defined bypositions [+3;+45] of exon 2 of the human GAA gene (SEQ ID NO:1) has alength from 10 to 100 nucleotides, preferably from 10 to 50 nucleotides,further preferably a length from 10 to 35 nucleotides, again furtherpreferably from 13 to 35 nucleotides or from 10 to 30 nucleotides, andhas, for example, a length of 10, or 15, or 20 or 30 nucleotides, andwherein further preferably said nucleotides are contiguous nucleotidesin said region defined by positions [+3;+45] of exon 2 of the human GAAgene (SEQ ID NO:1).

The present invention also relates to the nucleic acid as defined above,for use in a method for the treatment of Pompe disease. The patientstreated are those whose genome comprises a mutation preventing theinclusion of exon 2 of the GAA pre-mRNA within the mature GAA mRNA. Inparticular, the patient carries the c.-32 IVS1-13 T>G mutation or aIVS1.-3 C>N (wherein N may be A, T or G, in particular A or G) mutationin intron 1 of the gene coding GAA. Very preferably, the patient carriesthe c.-32-13 T>G mutation in intron 1 of the gene coding GAA. Typicallyand very preferred, the patient is a human patient and said humanpatient harbours at least one copy of the c.-32-13 T>G mutation in theGAA gene. Thus, in a further aspect, the present invention provides forthe inventive nucleic acid molecule, as defined herein, for use in amethod for the treatment of Pompe disease in a patient, wherein saidpatient is a human patient and said human patient harbours at least onecopy of the c.-32-13 T>G mutation in the GAA gene. Further preferably,said nucleic acid molecule comprises, or preferably consists of, anucleotide sequence complementary to a sequence comprised in the regiondefined by positions [+3;+45] of exon 2 of the GAA gene. In a verypreferred embodiment, the present invention provides for the inventivenucleic acid molecule, as defined herein, for use in a method for thetreatment of Pompe disease in a patient, wherein said patient is a humanpatient and said human patient harbours at least one copy of thec.-32-13 T>G mutation in the GAA gene, wherein preferably said nucleicacid molecule comprises, or preferably consists of, a nucleotidesequence complementary to a sequence comprised in the region defined bypositions [+3;+31] of exon 2 of the GAA gene, and wherein said nucleicacid molecule is an antisense oligonucleotide.

In a very preferred embodiment, the nucleic acid of the inventioncomprises or consists of the nucleotide sequence shown in SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16 or SEQ ID NO:17.

The invention further relates to a method for treating Pompe diseasecaused by non inclusion of exon 2, in particular Pompe disease caused bythe c.-32 IVS1-13 T>G mutation in the GAA gene, in a patient in needthereof, comprising the step of administering to said patient a nucleicacid as defined above. The nucleic acid improves inclusion of said exon2 during splicing.

This invention also relates to a method for correcting Pompe diseasecaused by a mutation preventing the inclusion of exon 2 of the GAApre-mRNA within the mature GAA mRNA, in particular the c.-32 IVS1-13 T>Gmutation, by using in vivo delivery of either gene vectors encodingengineered snRNAs such as U1 or U7 snRNAs harbouring the antisensesequences provided herein, or engineered autologous myogenic cells, withsaid optimized snRNAs, such as mesangioblasts, AC 133+ cells or anymesenchymal stem cells for correction of the glycogen storagedysfunction.

The invention further relates to a pharmaceutical composition comprisingthe nucleic acid of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Throughout the specification, several terms are employed and are definedin the following paragraphs as well as throughout the specification.

The terms “splicing” and “exon inclusion” are known to the skilledperson in the art, and used herein accordingly. The term “splicing”, asused herein, refer to the modification of a pre-mRNA followingtranscription, in which introns are removed and exons are joined. Theterm “exon inclusion”, as used herein, refers to the process leading tothe inclusion into the fully-processed mRNA of an exon which would havebeen otherwise left out of the mature mRNA due to a splicing defect.

The term “antisense oligonucleotide” refers to a single strand of DNA orRNA that is complementary to a chosen sequence. An antisenseoligonucleotide is capable of hybridizing to a pre-mRNA or an mRNAhaving a complementary coding or non coding nucleotide sequence.

The term “complementary”, as used herein, refers to a nucleic acidsequence that can form hydrogen bond(s) with another nucleic acidsequence by either traditional Watson-Crick base pairing or othernon-traditional types of pairing (e.g., Hoogsteen or reversed Hoogsteenhydrogen bonding) between complementary nucleosides or nucleotides.

In reference to the nucleic acid molecules of the present invention, andin particular in reference to the nucleic acid molecules of the presentinvention being antisense oligonucleotides, the binding free energy foran inventive nucleic acid molecule with its complementary sequence issufficient to allow the relevant function of the inventive nucleic acidmolecule to proceed and there is a sufficient degree of complementarityto avoid non-specific binding of the inventive nucleic acid molecule tonon-target sequences under conditions in which specific binding isdesired, i.e., under physiological conditions in the case of ex vivo orin vivo therapeutic treatment. Determination of binding free energiesfor nucleic acid molecules is well known in the art (see e.g., Turner etal., CSH Symp Quant Biol, 1987, LI/:123-133; Freier et al., Proc NatAcad Sci USA 1986, 83:9373-9377; and Turner et al., J Am Chem Soc, 1987,109:3783-3785). Thus, “complementary”, as used herein, indicate asufficient degree of complementarity or precise pairing such that stableand specific binding occurs between the inventive nucleic acid moleculeand the target nucleotide sequence of the pre-mRNA.

It is understood in the art that a nucleic acid molecule need not be100% complementary to a target nucleic acid sequence to becomplementary. That is, two or more nucleic acid molecules may be lessthan fully complementary. Complementarily is indicated by a percentageof contiguous residues in a nucleic acid molecule that can form hydrogenbonds with a second nucleic acid molecule. For example, if a firstnucleic acid molecule has 10 nucleotides and a second nucleic acidmolecule has 10 nucleotides, then base pairing of 5, 6, 7, 8, 9, or 10nucleotides between the first and second nucleic acid moleculesrepresents 50%, 60%, 70%, 80%, 90%, and 100% complementarity,respectively. “Fully” complementary nucleic acid molecules means thosein which all the contiguous residues of a first nucleic acid moleculewill hydrogen bond with the same number of contiguous residues in asecond nucleic acid molecule, wherein the nucleic acid molecules eitherboth have the same number of nucleotides (i.e., have the same length) orthe two molecules have different lengths. One of ordinary skill in theart would recognize that the inventive nucleic acid molecule providedherein is at least 80%, preferably at least 85%, further preferably atleast 90%, again further preferably at least 93%, again furtherpreferably at least 96%, again further preferably at least 98%, and mostpreferably 100% complementary to the target nucleotide sequence of thepre-mRNA.

Thus, the inventive nucleic acid molecule and the target nucleotidesequence of the pre-mRNA are complementary to each other when asufficient number of corresponding positions in each molecule areoccupied by nucleobases that can bond with each other to allow stableassociation between the inventive nucleic acid molecule and the targetnucleotide sequence of the pre-mRNA as indicated above. One skilled inthe art recognizes that the inclusion of mismatches is possible withouteliminating the ability of the oligomeric compounds to remain inassociation. Therefore, described herein are inventive nucleic acidmolecules, preferably being antisense oligonucleotides, that maycomprise up to about 20% nucleotides that are mismatched (i.e., are notnucleobase complementary to the corresponding nucleotides of thetarget). Preferably the inventive nucleic acid molecules, preferablybeing antisense oligonucleotides, contain no more than about 15%, morepreferably not more than about 10%, most preferably not more than 5% orno mismatches.

The term “acid alpha-glucosidase” (also called: lysosomalalpha-glucosidase or α-1,4-glucosidase) has its general meaning in theart and refers to a protein (e.g. enzyme) that in humans is encoded bythe GAA gene. Errors in this gene cause glycogen storage disease type II(Pompe disease). The GAA enzyme is essential for the degradation ofglycogen to glucose in lysosomes.

The terms “c.-32 IVS1-13 T>G” and “c.-32-13 T>G” are interchangeablyused herein, and refer to the same mutation in the GAA gene (den DunnenJ T, et al.; Hum Genet. 2001; 109:121-124).

In the context of the invention, the term “patient” refers to anysubject, afflicted with Pompe disease and harbouring at least one copyof the GAA gene comprising a mutation preventing inclusion of exon 2 inthe mRNA. In particular, the patient harbours at least one copy of thec.-32 IVS1-13 T>G mutation or of a IVS1.-3 C>N (wherein N may be A, T orG, in particular A or G) mutation in the GAA gene. Further preferred,the patient harbours at least one copy of the c.-32-13 T>G mutation inthe GAA gene. Typically and very preferred, the patient is a humanpatient and said human patient harbours at least one copy of thec.-32-13 T>G mutation in the GAA gene.

In its broadest meaning, the term “treating” or “treatment” refers toreversing, alleviating, inhibiting the progress of, or preventing thedisorder or condition to which such term applies, or one or moresymptoms of such disorder or condition.

“Pharmaceutically” or “pharmaceutically acceptable” refers to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to a mammal, especially ahuman, as appropriate. A pharmaceutically acceptable carrier orexcipient refers to a non-toxic solid, semi-solid or liquid filler,diluent, encapsulating material or formulation auxiliary of any type.

The inventors herein show that splice-switching strategies can be usedfor the treatment of patients with Pompe disease. In particular, asignificant subset of patients with Pompe disease, particularly thoseharbouring at least one copy of the c.-32 IVS1-13 T>G mutation, may betreated thanks to the present invention. The inventors demonstrateunprecedented efficient GAA restoration in cells from patients treatedex vivo with antisense oligomers targeting key domains of the GAApre-mRNA for rescuing inclusion of exon 2 in the mature mRNA.

Those of skill in the art will recognize that there are many ways todetermine or measure a level of functionality of a protein, and todetermine a level of increase or decrease of functionality e.g. inresponse to a treatment protocol. Such methods include but are notlimited to measuring or detecting an activity of the protein, etc. Suchmeasurements are generally made in comparison to a standard or controlor “normal” sample. In addition, when the protein's lack is involved ina disease process, disease symptoms may be monitored and/or measured inorder to indirectly detect the presence or absence of a correctlyfunctioning protein, or to gauge the success of a treatment protocolintended to remedy the lack of the protein. Particularly, the rescue ofGAA in cells from patients with Pompe disease (e.g. c.-32 IVS1-13 T>Gmutation or an IVS1.-3 C>N mutation) can be measured by several methodsrecognized. For example, by using RT-PCR for assessing the presence ofthe full length GAA mRNA (i.e. including exon 2), or testing GAAactivity by measuring levels of glycogen deposits in cells.

As will be understood by those of skill in the art, in the cell nucleus,eukaryotic genes are transcribed into pre-messenger RNAs (pre-mRNA),which contain both exons and introns. To form mature mRNA, splicingoccurs at specific sequences at the borders of exons and introns (splicesites) thereby removing introns and connecting exons to one another toform mRNA, which is translated into protein. During the two lastdecades, splice-switching approaches have been developed to interferewith such mechanisms. In particular, successful inclusion of exon 7 ofthe SMN2 gene has been reported by using antisense oligonucleotidesmasking an intronic splice silencer (ISS) spanning from nucleotide 10 to25 in intron 7. However, no information in the prior art is availableindicating that inclusion of exon 2 in the GAA RNA could be obtained bytargeting ESS sequences in said exon 2.

In the present invention, the protein that is stabilized or restored tofunction is the acid alpha-glucosidase (GAA). More specifically,exon-encoded sequences are force-included using an antisenseoligonucleotide (AON). In this case, an AON is designed to complementsuitable sequences, in particular RNA sequences within the pre-mRNAmolecule, which lower correct splicing of the targeted exon(s), therebyblocking sequences that may harbour negative CIS-elements, such asintronic splice silencers (ISS) or exonic splice silencers (ESS), whichwould exclude the targeted exon(s) into mature mRNA.

In the present invention, AON sequences are selected so as to bespecific, i.e. the AON's are complementary only to the sequences of thetargeted pre-mRNA and not to other nucleic acid sequences. The AON'sused in the practice of the invention may be of any suitable type, e.g.oligodeoxyribonucleotides, oligoribonucleotides, morpholinos,tricyclo-DNA-antisense oligonucleotides, tricyclo-phosphorothioate DNA,LNA, U7- or U1-modified AONs or conjugate products thereof such aspeptide-conjugated or nanoparticle-complexed AONs, which are known tothe skilled person in the art (Bell, N M, et al., ChemBioChem, 2009,10:2691-2703 and references cited therein). AONs employed in thepractice of the invention are generally from about 10 to about 35nucleotides in length, in particular from about 13 to about 35nucleotides or from about 10 to about 30 nucleotides, and may be forexample, about 10, or about 15, or about 20 or about 30 nucleotides ormore in length. Typically, morpholino-AONs are about 25-30 nucleotideslong, 2′PMO-AONs are about 20-25 nucleotides long, and tricyclo-AONs areabout 13-20 nucleotides long, U7 and U1-modified AONs may possibly carrylonger antisense sequences of about 50 nucleotides.

In particular object of this invention relates to a nucleic acidmolecule as described herein, linked to a modified U7 or U1 smallnuclear RNA (snRNA). Information on U7 modification can in particular befound in Goyenvalle A, et al., Science 2004, 306:1796-1799; GoyenvalleA, et al., Mol Ther, 2009 7:1234-1240; Denti M A, et al., Hum Gene Ther,2006 17(5):565-574; Gedicke-Hornung C, et. al., EMBO Mol Med, 20135(7):1060-1077; Hoogaars W M, et al., Hum Gene Ther, 201223(12):1269-1279, Vulin A, et al., Mol Ther, 2012 20(11):2120-2133;Goyenvalle A, et al., Hum Mol Genet, 2012 21(11):2559-2571; GoyenvalleA, et al., Mol Ther, 2012 20(6):1212-1221, Francois V, et al., NatStruct Mol Biol, 2011 18(1):85-87; Quenneville S P, et al., Mol Ther,2007 15(2):431-438; WO11113889; WO06021724; and references citedtherein. The various modifications of snRNA of U1 or U7, preferably ofU7, useful for the present invention are known for the skilled person inthe art and, by way of example, some of them are exemplified in thegiven literature.

U7 snRNA is normally involved in histone pre-mRNA 3′-end processing, butcan be converted into a versatile tool for splicing modulation by asmall change in the binding site for Sm/Lsm proteins. The antisensesequence embedded into a snRNP particle is therefore protected fromdegradation and accumulates in the nucleus where splicing occurs.

The terms “engineered Ux snRNA” or “modified Ux snRNA”, asinterchangeably used herein, refer to (i) the optimizing of the promoterdriving the transcription of the engineered Ux snRNA, wherein optimizingcan be enhancing or reducing the activity of the promoter, typically andpreferably enhancing the activity of the promoter; (ii) optimizing the“sm” domain of the natural snRNA, wherein optimizing encompasses, asindicated herein and in the cited references, the proper subcellularlocalization where splicing occurs; (iii) the natural antisense domainof the Ux snRNA is replaced by the “on purpose” antisense sequence, thuscapable to anneal the targeted pre-mRNA, and thus by the preferrednucleic acid molecules of the present invention.

Thus, in a preferred embodiment of the present invention, said nucleicacid molecule is linked to a modified Ux snRNA, preferably to a modifiedU7 or U1 snRNA, and further preferably said nucleic acid molecule islinked to a modified U7 snRNA, and again further preferably said nucleicacid molecule is linked to a modified U7SmOPT snRNA, wherein the SmOPTsequence is provided in FIG. 7 and refers to position 30 to 40 of SEQ IDNO: 18.

Thus, in a further preferred embodiment of the present invention, saidnucleic acid molecule useful for treating Pompe disease, wherein saidnucleic acid molecule comprises or consists of an antisenseoligonucleotide, wherein said antisense oligonucleotide is complementaryto a nucleotide sequence of the human acid alpha-glucosidase (GAA)pre-mRNA, wherein said nucleotide sequence of the GAA pre-mRNA is asequence comprised in the region defined by positions [|3;|31] of exon 2of the human GAA gene (SEQ ID NO:1), and wherein said sequence comprisedin the region defined by positions [+3;+31] of exon 2 of the human GAAgene (SEQ ID NO:1) has a length from 10 to 29 nucleotides, and whereinsaid nucleic acid molecule is linked to a modified U7 or U1 snRNA, andwherein preferably said nucleic acid molecule is linked to a modified U7snRNA.

In an again further preferred embodiment of the present invention, saidnucleic acid molecule useful for treating Pompe disease, wherein saidnucleic acid molecule comprises or consists of an antisenseoligonucleotide, wherein said antisense oligonucleotide is complementaryto a nucleotide sequence of the human acid alpha-glucosidase (GAA)pre-mRNA, wherein said nucleotide sequence of the GAA pre-mRNA is asequence comprised in the region defined by positions [+3;+31] of exon 2of the human GAA gene (SEQ ID NO:1), and wherein said sequence comprisedin the region defined by positions [+3;+31] of exon 2 of the human GAAgene (SEQ ID NO:1) has a length from 10 to 29 nucleotides, and whereinsaid nucleic acid molecule is linked to a modified U7 snRNA, whereinpreferably said modified modified U7 snRNA is a modified U7SmOPT snRNA,wherein the SmOPT sequence refers to position 30 to 40 of SEQ ID NO: 18.

In another preferred embodiment of the present invention, said nucleicacid molecule is able to restore the normal splicing of exon 2 of thehuman acid alpha-glucosidase (GAA) pre-mRNA, wherein said nucleic acidmolecule comprises or consists of an antisense oligonucleotide, whereinsaid antisense oligonucleotide is complementary to a nucleotide sequenceof said human acid alpha-glucosidase (GAA) pre-mRNA, wherein saidnucleotide sequence of said GAA pre-mRNA is a sequence comprised in theregion defined by positions [+3;+31] of exon 2 of the human GAA gene(SEQ ID NO:1), and wherein preferably said sequence comprised in theregion defined by positions [+3;+31] of exon 2 of the human GAA gene(SEQ ID NO:1) has a length from about 10 to about 29 nucleotides,preferably from about 13 to about 29 nucleotides, and has, for example,a length of about 10, or about 15, or about 20 or about 25 nucleotides.The term “able to restore the normal splicing of exon 2 of the humanacid alpha-glucosidase (GAA) pre-mRNA”, as used and described in detailherein, refers to the correct splicing of intron 1 allowing theformation of a complete GAA mRNA, which can be translated into a fullyfunctional protein (ATG starting codon is in exon 2: missing exon 2makes that the delta2-mRNA cannot be translated into human acidalpha-glucosidase).

In another preferred embodiment of the present invention, said nucleicacid molecule is able to restore the normal splicing of exon 2 of thehuman acid alpha-glucosidase (GAA) pre-mRNA displaying the c.-32-13T>Gmutation, wherein said nucleic acid molecule comprises or consists of anantisense oligonucleotide, wherein said antisense oligonucleotide iscomplementary to a nucleotide sequence of said human acidalpha-glucosidase (GAA) pre-mRNA, wherein said nucleotide sequence ofsaid GAA pre-mRNA is a sequence comprised in the region defined bypositions [+3;+31] of exon 2 of the human GAA gene (SEQ ID NO:1), andwherein preferably said sequence comprised in the region defined bypositions [+3;+31] of exon 2 of the human GAA gene (SEQ ID NO:1) has alength from about 10 to about 29 nucleotides, preferably from about 13to about 29 nucleotides, and has, for example, a length of about 10, orabout 15, or about 20 or about 25 nucleotides.

In again another aspect, the present invention provides for a nucleicacid molecule is able to restore the normal splicing of exon 2 of thehuman acid alpha-glucosidase pre-mRNA displaying the c.-32-13T>Gmutation, wherein said nucleic acid molecule comprises or consists of anantisense oligonucleotide, wherein said antisense oligonucleotide iscomplementary to a nucleotide sequence of the human acidalpha-glucosidase (GAA) pre-mRNA, wherein said nucleotide sequence ofthe GAA pre-mRNA is a sequence comprised in the region defined bypositions [+3;+31] of exon 2 of the human GAA gene (SEQ ID NO:1), andwherein said sequence comprised in the region defined by positions[+3;+31] of exon 2 of the human GAA gene (SEQ ID NO:1) has a length from10 to 29 nucleotides, and wherein said nucleic acid molecule is linkedto a modified U7 or U1 snRNA, and wherein preferably said nucleic acidmolecule is linked to a modified U7 snRNA.

In again another aspect, the present invention provides for a nucleicacid molecule is able to restore the normal splicing of exon 2 of thehuman acid alpha-glucosidase pre-mRNA displaying the c.-32-13T>Gmutation, wherein said nucleic acid molecule comprises or consists of anantisense oligonucleotide, wherein said antisense oligonucleotide iscomplementary to a nucleotide sequence of the human acidalpha-glucosidase (GAA) pre-mRNA, wherein said nucleotide sequence ofthe GAA pre-mRNA is a sequence comprised in the region defined bypositions [+3;+31] of exon 2 of the human GAA gene (SEQ ID NO:1), andwherein said sequence comprised in the region defined by positions[+3;+31] of exon 2 of the human GAA gene (SEQ ID NO:1) has a length from10 to 29 nucleotides, and wherein said nucleic acid molecule is linkedto a modified U7 snRNA, wherein preferably said modified modified U7snRNA is a modified U7SmOPT snRNA, wherein the SmOPT sequence refers toposition 30 to 40 of SEQ ID NO: 18.

In a further aspect, the present invention provides for a vectorcomprising the nucleic acid molecule of the invention. Preferably, saidvector comprises a nucleic acid molecule able to restore the normalsplicing of exon 2 of the human acid alpha-glucosidasc pre-mRNAdisplaying the c.-32-13T>G mutation, wherein said nucleic acid moleculecomprises or consists of an antisense oligonucleotide, wherein saidantisense oligonucleotide is complementary to a nucleotide sequence ofthe human acid alpha-glucosidase (GAA) pre-mRNA, wherein said nucleotidesequence of the GAA pre-mRNA is a sequence comprised in the regiondefined by positions [+3;+31] of exon 2 of the human GAA gene (SEQ IDNO:1), and wherein said sequence comprised in the region defined bypositions [+3;+31] of exon 2 of the human GAA gene (SEQ ID NO:1) has alength from 10 to 29 nucleotides, and wherein said nucleic acid moleculeis linked to a modified U7 snRNA, wherein preferably said modifiedmodified U7 snRNA is a modified U7SmOPT snRNA, wherein the SmOPTsequence refers to position 30 to 40 of SEQ ID NO: 18. Furtherpreferably, said vector is selected from plasmids, adenoviral vectors,associated-adenoviral vectors and lentiviral vectors, preferably fromadenoviral vectors, associated-adenoviral vectors. Thus, in a verypreferred embodiment, said vector is an adenoviral vector or anassociated-adenoviral vector and comprises a nucleic acid molecule ableto restore the normal splicing of exon 2 of the human acidalpha-glucosidase pre-mRNA displaying the c.-32-13T>G mutation, whereinsaid nucleic acid molecule comprises or consists of an antisenseoligonucleotide, wherein said antisense oligonucleotide is complementaryto a nucleotide sequence of the human acid alpha-glucosidase (GAA)pre-mRNA, wherein said nucleotide sequence of the GAA pre-mRNA is asequence comprised in the region defined by positions [+3;+31] of exon 2of the human GAA gene (SEQ ID NO:1), and wherein said sequence comprisedin the region defined by positions [+3;+31] of exon 2 of the human GAAgene (SEQ ID NO:1) has a length from 10 to 29 nucleotides, and whereinsaid nucleic acid molecule is linked to a modified U7 snRNA, whereinsaid modified U7 snRNA is a modified U7SmOPT snRNA, wherein the SmOPTsequence refers to position 30 to 40 of SEQ ID NO: 18.

In another aspect, the present invention provides for an isolatedeukaryotic cell transfected by the inventive vector, and wherein saidcell is preferably a skeletal muscle cell, a myoblast or a cell capableof muscle differentiation.

In another aspect, the present invention provides for a pharmaceuticalcomposition comprising the inventive vector or the inventive cell. In afurther aspect, the present invention provides for the inventive vectoror the inventive cell as a medicament. In again another aspect, thepresent invention provides for the inventive vector for treating Pompedisease. In another aspect, the present invention provides for theinventive cell for treating Pompe disease. In again another aspect, thepresent invention provides for a method of restoring the function ofhuman acid alpha-glucosidasc by inclusion of exon 2 comprising the stepof contacting a cell with the inventive vector.

An object of the invention relates to a nucleic acid moleculecomplementary to a nucleic acid sequence 5′ in exon 2 of a GAA gene, thenucleic acid molecule being able to set free the acceptor splice site ofexon 2 from a strong hairpin formed when the GAA gene comprises a pointmutation resulting in exon 2 exclusion from the mRNA such as the c.-32IVS1-13 T>G or an IVS1.-3 C>N mutation.

Another object of the invention relates to a nucleic acid moleculecomplementary to a nucleic acid sequence of a GAA gene, this nucleicacid molecule being able to correct splicing of exon 2 of the GAApre-mRNA. The present invention relates in particular to a nucleic acidmolecule (or otherwise referred to as an antisense oligonucleotide inthe present application) complementary to exon 2 of a GAA gene, inparticular to a 5′ region of exon 2, said nucleic acid moleculecomprising a nucleotide sequence complementary to one or more ExonicSilencer Sequence(s) (ESS) in exon 2 of the gene coding for acidalpha-glucosidase. Said nucleic acid molecule is thus capable of maskingthe ESSs in exon 2 and therefore promotes inclusion of said exon in themRNA coding acid alpha-glucosidase. ESS sequences recruit splicinginhibitors on the pre-mRNA. Masking of these sequences may thereforedecrease this recruitment and promote inclusion of exon 2 in the mRNA.ESS may be found in a given sequence searching for consensus sequences.In particular, one may use the Human Splicing Finder bioinformatics toolin identifying such ESS (Desmet et al., 2009, Human Splicing Finder: anonline bioinformatics tool to predict splicing signals, Nucleic AcidsResearch, 2009, 1-14; www.umd.be/HSF/).

The present inventors show that a nucleic acid molecule of theinvention, which is a molecule targeting one or several ESS in exon 2,is able to restore inclusion of said exon 2 in the mRNA of patientscarrying a mutation of the GAA that would result in the exclusion ofsaid exon 2 in the absence of the nucleic acid molecule of theinvention.

Therefore, in an embodiment of the present invention, the nucleic acidmolecule is 10 to 50 nucleotides in length, and complementary to anucleotide sequence of the acid alpha-glucosidase pre-mRNA, wherein saidnucleic acid is able to correct the splicing of exon 2 of the acidalpha-glucosidase pre-mRNA, and wherein said nucleic acid moleculecomprises a nucleotide sequence complementary to the Exonic SilencerSequence (ESS) in exon 2 of the gene coding for acid alpha-glucosidase.

In a particular embodiment, the nucleic acid molecule of the inventioncomprises a nucleotide sequence complementary to a sequence comprised inthe region defined by positions +3+45 of exon 2 of the GAA gene. In afurther particular embodiment, the nucleic acid molecule of theinvention comprises a nucleotide sequence complementary to a sequencecomprised in the region defined by positions [+3;+31] of exon 2 of thehuman GAA gene. In a particular embodiment, the nucleic acid molecule ofthe invention comprises or consists of a nucleotide sequencecomplementary to one or more of the sequences defined by positions 3-17,7-21 or 17-31 in SEQ ID NO:1 or in a sequence having at least 90%homology with SEQ ID NO:1, in particular at least 95%, more particularlyat least 99%. In a further preferred embodiment of the presentinvention, the nucleic acid molecule comprises or consists of anucleotide sequence complementary to one or more of the sequencesselected from (i) the sequence defined by position [+3;+17] of SEQ IDNO:1; (ii) the sequence defined by position [+7;+21] of SEQ ID NO:1;(iii) the sequence defined by position [+17;+31] of SEQ ID NO:1; (iv) asequence having at least 90% homology, preferably at least 99% homologywith the sequence defined in either (i), (ii) or (iii).

In a more particular embodiment, said nucleic acid molecule iscomplementary to the nucleotide sequence shown in SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:12 or SEQ ID NO:13.

SEQ ID NO: 2 5′-CUGUAGGAGCUGUCC-3′ SEQ ID NO: 3 5′-AGGAGCUGUCCAGGC-3′SEQ ID NO: 4 5′-CAGGCCAUCUCCAAC-3′ SEQ ID NO: 55′-CUGUAGGAGCUGUCCAGGCCAUCUCCAACCAUGGGAGUGAGGCA-3′ SEQ ID NO: 125′-CUGUAGGAGCUGUCCAGGCCAUCUCCAAC-3′ SEQ ID NO: 135′-CUGUAGGAGCUGUCCAGGCCAUCUCCAACCAUGGGAGUGAGGC-3′

The antisense oligonucleotides (AONs) of the invention can besynthesized de novo using any of a number of procedures well known inthe art. For example, the b-cyanoethyl phosphoramidite method (Beaucage,S. L., and Caruthers, M. H., Tet. Let. 22:1859, 1981); nucleosideII-phosphonate method (Garegg et al., Tet. Let. 27:4051-4054, 1986;Froehler et al., Nucl. Acid. Res. 14:5399-5407, 1986; Garegg et al.,Tet. Let. 27:4055-4058, 1986, Gaffney et al., Tet. Let. 29:2619-2622,1988). These chemistries can be performed by a variety of automatednucleic acid synthesizers available in the market. These nucleic acidsmay be referred to as synthetic nucleic acids. Alternatively, AON's canbe produced on a large scale in plasmids, (see Sambrook, T., et al.,“Molecular Cloning: A Laboratory Manual”, Cold Spring Harbor laboratoryPress, New York, 1989). AON's can be prepared from existing nucleic acidsequences using known techniques, such as those employing restrictionenzymes, exonucleases or endonucleases. AON's prepared in this mannermay be referred to as isolated nucleic acids.

For use in vivo, the AONs may be stabilized. A “stabilized” AON refersto an AON that is relatively resistant to in vivo degradation (e.g. viaan exo- or endo-nuclease). Stabilization can be a function of length orsecondary structure. Alternatively, AON stabilization can beaccomplished via phosphate backbone modifications. Preferred stabilizedAON's of the instant invention have a modified backbone, e.g. havephosphorothioate linkages to provide maximal activity and protect theAON from degradation by intracellular exo- and endo-nucleases. Otherpossible stabilizing modifications include phosphodiester modifications,combinations of phosphodiester and phosphorothioate modifications,methylphosphonate, methyl-phosphorothioate, phosphorodithioate,p-ethoxy, and combinations thereof. Chemically stabilized, modifiedversions of the AON's also include “Morpholinos” (phosphorodiamidatemorpholino oligomers, PMOs), 2′-O-Met oligomers, tricyclo (tc)-DNAoligomers (WO2010/115993), tricyclo(tc)-phosphorothioate DNA oligomers(WO2013/053928), LNAs etc, which are all known to the skilled person inthe art (Bell, N M, et al., ChemBioChem, 2009, 10:2691-2703 andreferences cited therein).

The term “tricyclo-DNA (tc-DNA)” refers to a class of constrainedoligodeoxyribonucleotide analogs in which each nucleotide is modified bythe introduction of a cyclopropane ring to restrict conformationalflexibility of the backbone and to optimize the backbone geometry of thetorsion angle γ as (Ittig D, et al., Nucleic Acids Res, 2004,32:346-353; Ittig D, et al., Prague, Academy of Sciences of the CzechRepublic. 1:21-26 (Coll. Symp. Series, Hoece, M., 2005); Ivanova et al.,Oligonucleotides 2007, 17:54-65; Renneberg D, et al., Nucleic Acids Res,2002, 15 30:2751-2757; Renneberg D, et al., Chembiochem, 2004,5:1114-1118; and Renneberg D, et al., JACS, 2002, 124:5993-6002). Indetail, the tc-DNA differs structurally from DNA by an additionalethylene bridge between the centers C(3′) and C(5′) of the nucleosides,to which a cyclopropane unit is fused for further enhancement ofstructural rigidity as shown in the following formula (“tc-nucleoside”):

The terms “phosphorothioate linkage” or “phosphorothioate modification”,as interchangeably used herein, refers to a 5′ . . . -0-P(S)-0- . . . 3′moiety between two adjacent nucleosides in a nucleic acid molecule.

Thus, the term “tricyclo-phosphorothioate DNA (tc-PS-DNA)” refers totc-DNA, wherein at least two adjacent tc-nucleosides, preferably morethan two adjacent tc-nucleosides, and most preferably alltc-nucleosides, are joined by inter nucleoside phosphorothioatelinkages. If other modifications are present they includephosphodiester, methylphosphonate, methyl-phosphorothioate,phosphorodithioate, and p-ethoxy modifications, and combinationsthereof.

In a further preferred embodiment, said nucleic acid molecule comprisesor consists of tricyclo-DNA-antisense oligonucleotides, and whereinpreferably said nucleic acid molecule comprises or consists of asequence selected from SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, andSEQ ID NO: 17.

In a further aspect, the present invention provides for a nucleic acidmolecule useful for treating Pompe disease comprising or consisting ofan antisense oligonucleotide, wherein said antisense oligonucleotide isa tc-DNA and is complementary to a nucleotide sequence of the human acidalpha-glucosidase (GAA) pre-mRNA, wherein said nucleotide sequence ofthe GAA pre-mRNA is a sequence comprised in the region defined bypositions [+3;+45] of exon 2 of the human GAA gene (SEQ ID NO:1), andwherein preferably said sequence comprised in the region defined bypositions [+3;+45] of exon 2 of the human GAA gene (SEQ ID NO:1) has alength from 10 to 35 nucleotides, preferably from 13 to 35 nucleotidesor from 10 to 30 nucleotides, and has, for example, a length of 10, or15, or 20 or 30 nucleotides, and wherein further preferably saidnucleotides are contiguous nucleotides in said region defined bypositions [+3;+45] of exon 2 of the human GAA gene (SEQ ID NO:1).Preferably said nucleic acid molecule comprises or consists of asequence selected from SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, andSEQ ID NO: 17.

In a further aspect, the present invention provides for a nucleic acidmolecule useful for treating Pompe disease comprising or consisting ofan antisense oligonucleotide, wherein said antisense oligonucleotide isa tc-DNA and is complementary to a nucleotide sequence of the human acidalpha-glucosidase (GAA) pre-mRNA, wherein said nucleotide sequence ofthe GAA pre-mRNA is a sequence comprised in the region defined bypositions [+3;+31] of exon 2 of the human GAA gene (SEQ ID NO:1), andwherein preferably said sequence comprised in the region defined bypositions [+3;+31] of exon 2 of the human GAA gene (SEQ ID NO:1) has alength from about 10 to about 29 nucleotides, preferably from about 13to about 29 nucleotides, and has, for example, a length of about 10, orabout 15, or about 20 or about 25 nucleotides, and wherein furtherpreferably said nucleotides are contiguous nucleotides in said regiondefined by positions [+3;+31] of exon 2 of the human GAA gene (SEQ IDNO:1). Preferably said nucleic acid molecule comprises or consists of asequence selected from SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, andSEQ ID NO: 17.

In a further aspect, the present invention provides for a nucleic acidmolecule able to correct the splicing of exon 2 of the human acidalpha-glucosidase pre-mRNA, wherein said nucleic acid molecule comprisesor consists of an antisense oligonucleotide, wherein said antisenseoligonucleotide is a tc-DNA and is complementary to a nucleotidesequence of the human acid alpha-glucosidase (GAA) pre-mRNA, whereinsaid nucleotide sequence of the GAA pre-mRNA is a sequence comprised inthe region defined by positions [+3;+45] of exon 2 of the human GAA gene(SEQ ID NO:1), and wherein preferably said sequence comprised in theregion defined by positions [+3;+45] of exon 2 of the human GAA gene(SEQ ID NO:1) has a length from 10 to 50 nucleotides, preferably from 10to 35 nucleotides, preferably from 13 to 35 nucleotides or from 10 to 30nucleotides, and has, for example, a length of 10, or 15, or 20 or 30nucleotides, and wherein further preferably said nucleotides arecontiguous nucleotides in said region defined by positions [+3;+45] ofexon 2 of the human GAA gene (SEQ ID NO:1). Preferably said nucleic acidmolecule comprises or consists of a sequence selected from SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17.

In a further aspect, the present invention provides for a nucleic acidmolecule able to correct the splicing of exon 2 of the human acidalpha-glucosidase pre-mRNA, wherein said nucleic acid molecule comprisesor consists of an antisense oligonucleotide, wherein said antisenseoligonucleotide is a tc-DNA and is complementary to a nucleotidesequence of the human acid alpha-glucosidase (GAA) pre-mRNA, whereinsaid nucleotide sequence of the GAA pre-mRNA is a sequence comprised inthe region defined by positions [+3;+31] of exon 2 of the human GAA gene(SEQ ID NO:1), and wherein preferably said sequence comprised in theregion defined by positions [+3;+31] of exon 2 of the human GAA gene(SEQ ID NO:1) has a length from 10 to 35 nucleotides, further preferablyfrom 13 to 35 nucleotides or from 10 to 30 nucleotides, and has, forexample, a length of 10, or 15, or 20 or 30 nucleotides, and whereinfurther preferably said nucleotides are contiguous nucleotides in saidregion defined by positions [+3;+31] of exon 2 of the human GAA gene(SEQ ID NO:1). Preferably said nucleic acid molecule comprises orconsists of a sequence selected from SEQ ID NO: 14, SEQ ID NO: 15, SEQID NO: 16, and SEQ ID NO: 17.

In an another aspect, the present invention provides for a nucleic acidmolecule able to restore the normal splicing of exon 2 of the human acidalpha-glucosidase pre-mRNA displaying the c.-32-13T>G mutation, whereinsaid nucleic acid molecule comprises or consists of an antisenseoligonucleotide, wherein said antisense oligonucleotide is a tc-DNA andis complementary to a nucleotide sequence of the human acidalpha-glucosidase (GAA) pre-mRNA, wherein said nucleotide sequence ofthe GAA pre-mRNA is a sequence comprised in the region defined bypositions [+3;+45] of exon 2 of the human GAA gene (SEQ ID NO:1), andwherein preferably said sequence comprised in the region defined bypositions [+3;+45] of exon 2 of the human GAA gene (SEQ ID NO:1) has alength from 10 to 35 nucleotides, preferably from 13 to 35 nucleotidesor from 10 to 30 nucleotides, and has, for example, a length of 10, or15, or 20 or 30 nucleotides, and wherein further preferably saidnucleotides are contiguous nucleotides in said region defined bypositions [+3;+45] of exon 2 of the human GAA gene (SEQ ID NO:1).Preferably said nucleic acid molecule comprises or consists of asequence selected from SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, andSEQ ID NO: 17.

In an another aspect, the present invention provides for a nucleic acidmolecule able to restore the normal splicing of exon 2 of the human acidalpha-glucosidase pre-mRNA displaying the c.-32-13T>G mutation, whereinsaid nucleic acid molecule comprises or consists of an antisenseoligonucleotide, wherein said antisense oligonucleotide is a tc-DNA andis complementary to a nucleotide sequence of the human acidalpha-glucosidase (GAA) pre-mRNA, wherein said nucleotide sequence ofthe GAA pre-mRNA is a sequence comprised in the region defined bypositions [+3;+31] of exon 2 of the human GAA gene (SEQ ID NO:1), andwherein preferably said sequence comprised in the region defined bypositions [+3;+31] of exon 2 of the human GAA gene (SEQ ID NO:1) has alength from about 10 to about 29 nucleotides, preferably from about 13to about 29 nucleotides, and has, for example, a length of about 10, orabout 15, or about 20 or about 25 nucleotides, and wherein furtherpreferably said nucleotides are contiguous nucleotides in said regiondefined by positions [+3;+31] of exon 2 of the human GAA gene (SEQ IDNO:1). Preferably said nucleic acid molecule comprises or consists of asequence selected from SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, andSEQ ID NO: 17.

In a further preferred embodiment, said nucleic acid molecule comprisesor consists of tricyclo-phosphorothioate DNA-antisense oligonucleotides,and wherein preferably said nucleic acid molecule comprises or consistsof a sequence selected from SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16,and SEQ ID NO: 17.

In a further aspect, the present invention provides for a nucleic acidmolecule useful for treating Pompe disease comprising or consisting ofan antisense oligonucleotide, wherein said antisense oligonucleotide isa tc-PS-DNA and is complementary to a nucleotide sequence of the humanacid alpha-glucosidase (GAA) pre-mRNA, wherein said nucleotide sequenceof the GAA pre-mRNA is a sequence comprised in the region defined bypositions [+3;+45] of exon 2 of the human GAA gene (SEQ ID NO:1), andwherein preferably said sequence comprised in the region defined bypositions [+3;+45] of exon 2 of the human GAA gene (SEQ ID NO:1) has alength from 10 to 35 nucleotides, preferably from 13 to 35 nucleotidesor from 10 to 30 nucleotides, and has, for example, a length of 10, or15, or 20 or 30 nucleotides, and wherein further preferably saidnucleotides are contiguous nucleotides in said region defined bypositions [+3;+45] of exon 2 of the human GAA gene (SEQ ID NO:1).Preferably said nucleic acid molecule comprises or consists of asequence selected from SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, andSEQ ID NO: 17. In further preferred embodiments, all tc-nucleosides ofsaid tc-PS-DNA are joined by inter nucleoside phosphorothioate linkages.

In a further aspect, the present invention provides for a nucleic acidmolecule useful for treating Pompe disease comprising or consisting ofan antisense oligonucleotide, wherein said antisense oligonucleotide isa tc-PS-DNA and is complementary to a nucleotide sequence of the humanacid alpha-glucosidase (GAA) pre-mRNA, wherein said nucleotide sequenceof the GAA pre-mRNA is a sequence comprised in the region defined bypositions [+3;+31] of exon 2 of the human GAA gene (SEQ ID NO:1), andwherein preferably said sequence comprised in the region defined bypositions [+3;+31] of exon 2 of the human GAA gene (SEQ ID NO:1) has alength from about 10 to about 29 nucleotides, preferably from about 13to about 29 nucleotides, and has, for example, a length of about 10, orabout 15, or about 20 or about 25 nucleotides, and wherein furtherpreferably said nucleotides are contiguous nucleotides in said regiondefined by positions [+3;+31] of exon 2 of the human GAA gene (SEQ IDNO:1). Preferably said nucleic acid molecule comprises or consists of asequence selected from SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, andSEQ ID NO: 17. In further preferred embodiments, all tc-nucleosides ofsaid tc-PS-DNA are joined by inter nucleoside phosphorothioate linkages.

In a further aspect, the present invention provides for a nucleic acidmolecule able to correct the splicing of exon 2 of the human acidalpha-glucosidase pre-mRNA, wherein said nucleic acid molecule comprisesor consists of an antisense oligonucleotide, wherein said antisenseoligonucleotide is a tc-PS-DNA and is complementary to a nucleotidesequence of the human acid alpha-glucosidase (GAA) pre-mRNA, whereinsaid nucleotide sequence of the GAA pre-mRNA is a sequence comprised inthe region defined by positions [+3;+45] of exon 2 of the human GAA gene(SEQ ID NO:1), and wherein preferably said sequence comprised in theregion defined by positions [+3;+45] of exon 2 of the human GAA gene(SEQ ID NO:1) has a length from 10 to 50 nucleotides, preferably from 10to 35 nucleotides, preferably from 13 to 35 nucleotides or from 10 to 30nucleotides, and has, for example, a length of 10, or 15, or 20 or 30nucleotides, and wherein further preferably said nucleotides arecontiguous nucleotides in said region defined by positions [+3;+45] ofexon 2 of the human GAA gene (SEQ ID NO:1). Preferably said nucleic acidmolecule comprises or consists of a sequence selected from SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17. In furtherpreferred embodiments, all tc-nucleosides of said tc-PS-DNA are joinedby inter nucleoside phosphorothioate linkages.

In a further aspect, the present invention provides for a nucleic acidmolecule able to correct the splicing of exon 2 of the human acidalpha-glucosidase pre-mRNA, wherein said nucleic acid molecule comprisesor consists of an antisense oligonucleotide, wherein said antisenseoligonucleotide is a tc-PS-DNA and is complementary to a nucleotidesequence of the human acid alpha-glucosidase (GAA) pre-mRNA, whereinsaid nucleotide sequence of the GAA pre-mRNA is a sequence comprised inthe region defined by positions [+3;+31] of exon 2 of the human GAA gene(SEQ ID NO:1), and wherein preferably said sequence comprised in theregion defined by positions [+3;+31] of exon 2 of the human GAA gene(SEQ ID NO:1) has a length from 10 to 35 nucleotides, further preferablyfrom 13 to 35 nucleotides or from 10 to 30 nucleotides, and has, forexample, a length of 10, or 15, or 20 or 30 nucleotides, and whereinfurther preferably said nucleotides are contiguous nucleotides in saidregion defined by positions [+3;+31] of exon 2 of the human GAA gene(SEQ ID NO:1). Preferably said nucleic acid molecule comprises orconsists of a sequence selected from SEQ ID NO: 14, SEQ ID NO: 15, SEQID NO: 16, and SEQ ID NO: 17. In further preferred embodiments, alltc-nucleosides of said tc-PS-DNA are joined by inter nucleosidephosphorothioate linkages.

In an another aspect, the present invention provides for a nucleic acidmolecule able to restore the normal splicing of exon 2 of the human acidalpha-glucosidase pre-mRNA displaying the c.-32-13T>G mutation, whereinsaid nucleic acid molecule comprises or consists of an antisenseoligonucleotide, wherein said antisense oligonucleotide is a tc-PS-DNAand is complementary to a nucleotide sequence of the human acidalpha-glucosidase (GAA) pre-mRNA, wherein said nucleotide sequence ofthe GAA pre-mRNA is a sequence comprised in the region defined bypositions [+3;+45] of exon 2 of the human GAA gene (SEQ ID NO:1), andwherein preferably said sequence comprised in the region defined bypositions [+3;+45] of exon 2 of the human GAA gene (SEQ ID NO:1) has alength from 10 to 35 nucleotides, preferably from 13 to 35 nucleotidesor from 10 to 30 nucleotides, and has, for example, a length of 10, or15, or 20 or 30 nucleotides, and wherein further preferably saidnucleotides are contiguous nucleotides in said region defined bypositions [+3;+45] of exon 2 of the human GAA gene (SEQ ID NO:1).Preferably said nucleic acid molecule comprises or consists of asequence selected from SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, andSEQ ID NO: 17. In further preferred embodiments, all tc-nucleosides ofsaid tc-PS-DNA are joined by inter nucleoside phosphorothioate linkages.

In an another aspect, the present invention provides for a nucleic acidmolecule able to restore the normal splicing of exon 2 of the human acidalpha-glucosidase pre-mRNA displaying the c.-32-13T>G mutation, whereinsaid nucleic acid molecule comprises or consists of an antisenseoligonucleotide, wherein said antisense oligonucleotide is a tc-PS-DNAand is complementary to a nucleotide sequence of the human acidalpha-glucosidase (GAA) pre-mRNA, wherein said nucleotide sequence ofthe GAA pre-mRNA is a sequence comprised in the region defined bypositions [+3;+31] of exon 2 of the human GAA gene (SEQ ID NO:1), andwherein preferably said sequence comprised in the region defined bypositions [+3;+31] of exon 2 of the human GAA gene (SEQ ID NO:1) has alength from about 10 to about 29 nucleotides, preferably from about 13to about 29 nucleotides, and has, for example, a length of about 10, orabout 15, or about 20 or about 25 nucleotides, and wherein furtherpreferably said nucleotides are contiguous nucleotides in said regiondefined by positions [+3;+31] of exon 2 of the human GAA gene (SEQ IDNO:1). Preferably said nucleic acid molecule comprises or consists of asequence selected from SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, andSEQ ID NO: 17. In further preferred embodiments, all tc-nucleosides ofsaid tc-PS-DNA are joined by inter nucleoside phosphorothioate linkages.

In a very preferred embodiment of the present invention, said nucleicacid molecule comprises or consists of a sequence as set forth in SEQ IDNO: 14.

In a further very preferred embodiment of the present invention, saidnucleic acid molecule comprises or consists of a sequence as set forthin SEQ ID NO: 15.

In an again a further very preferred embodiment of the presentinvention, said nucleic acid molecule comprises or consists of asequence as set forth in SEQ ID NO: 16.

In a further very preferred embodiment of the present invention, saidnucleic acid molecule comprises or consists of a sequence as set forthin SEQ ID NO: 17.

In a very preferred embodiment of the present invention, said nucleicacid molecule comprises or consists of a sequence as set forth in SEQ IDNO: 14, wherein said nucleic acid molecule is an antisenseoligonucleotide, and wherein said antisense oligonucleotide is a tc-DNA.

In a further very preferred embodiment of the present invention, saidnucleic acid molecule comprises or consists of a sequence as set forthin SEQ ID NO: 15, wherein said nucleic acid molecule is an antisenseoligonucleotide, and wherein said antisense oligonucleotide is a tc-DNA.

In an again a further very preferred embodiment of the presentinvention, said nucleic acid molecule comprises or consists of asequence as set forth in SEQ ID NO: 16, wherein said nucleic acidmolecule is an antisense oligonucleotide, and wherein said antisenseoligonucleotide is a tc-DNA.

In a further very preferred embodiment of the present invention, saidnucleic acid molecule comprises or consists of a sequence as set forthin SEQ ID NO: 17, wherein said nucleic acid molecule is an antisenseoligonucleotide, and wherein said antisense oligonucleotide is a tc-DNA.

In a very preferred embodiment of the present invention, said nucleicacid molecule comprises or consists of a sequence as set forth in SEQ IDNO: 14, wherein said nucleic acid molecule is an antisenseoligonucleotide, and wherein said antisense oligonucleotide is atc-PS-DNA, wherein all tc-nucleosides of said tc-PS-DNA are joined byinter nucleoside phosphorothioate linkages.

In a further very preferred embodiment of the present invention, saidnucleic acid molecule comprises or consists of a sequence as set forthin SEQ ID NO: 15, wherein said nucleic acid molecule is an antisenseoligonucleotide, and wherein said antisense oligonucleotide is atc-PS-DNA, wherein all tc-nucleosides of said tc-PS-DNA are joined byinter nucleoside phosphorothioate linkages.

In an again a further very preferred embodiment of the presentinvention, said nucleic acid molecule comprises or consists of asequence as set forth in SEQ ID NO: 16, wherein said nucleic acidmolecule is an antisense oligonucleotide, and wherein said antisenseoligonucleotide is a tc-PS-DNA, wherein all tc-nucleosides of saidtc-PS-DNA are joined by inter nucleoside phosphorothioate linkages.

In a further very preferred embodiment of the present invention, saidnucleic acid molecule comprises or consists of a sequence as set forthin SEQ ID NO: 17, wherein said nucleic acid molecule is an antisenseoligonucleotide, and wherein said antisense oligonucleotide is atc-PS-DNA, wherein all tc-nucleosides of said tc-PS-DNA are joined byinter nucleoside phosphorothioate linkages.

In a very preferred embodiment of the present invention, said nucleicacid molecule comprises or consists of a sequence as set forth in SEQ IDNO: 14, wherein all thymines (t) of said SEQ ID NO: 14 are substitutedby uracils (u), wherein said nucleic acid molecule is an antisenseoligonucleotide, and wherein said antisense oligonucleotide is a2′-O-methyl-oligoribonucleotide. Thus, preferably, said nucleic acidmolecule comprises or consists of a sequence as set forth in SEQ ID NO:14, wherein all thymines (t) of said SEQ ID NO: 14 are substituted byuracils (u), wherein said nucleic acid molecule is a 2′-O-methyl-RNAwith full phosphorothioate backbone antisense oligonucleotides(2′-O-Met). Thus, in a very preferred embodiment of the presentinvention, said nucleic acid molecule comprises or consists of asequence as set forth in SEQ ID NO: 14, wherein all thymines (t) of saidSEQ ID NO: 14 are substituted by uracils (u), wherein said nucleic acidmolecule is an antisense oligonucleotide, and wherein said antisenseoligonucleotide is a 2′-O-methyl-phosphorothioate oligoribonucleotide.

In a further very preferred embodiment of the present invention, saidnucleic acid molecule comprises or consists of a sequence as set forthin SEQ ID NO: 15, wherein all thymines (t) of said SEQ ID NO: 15 aresubstituted by uracils (u), wherein said nucleic acid molecule is anantisense oligonucleotide, and wherein said antisense oligonucleotide isa 2′-O-methyl-oligoribonucleotide. Thus, preferably, said nucleic acidmolecule comprises or consists of a sequence as set forth in SEQ ID NO:15, wherein all thymines (t) of said SEQ ID NO: 15 are substituted byuracils (u), wherein said nucleic acid molecule is a 2′-O-methyl-RNAwith full phosphorothioate backbone antisense oligonucleotides(2′-O-Met). Thus, in a further very preferred embodiment of the presentinvention, said nucleic acid molecule comprises or consists of asequence as set forth in SEQ ID NO: 15, wherein all thymines (t) of saidSEQ ID NO: 15 are substituted by uracils (u), wherein said nucleic acidmolecule is an antisense oligonucleotide, and wherein said antisenseoligonucleotide is a 2′-O-methyl-phosphorothioate oligoribonucleotide.

In an again a further very preferred embodiment of the presentinvention, said nucleic acid molecule comprises or consists of asequence as set forth in SEQ ID NO: 16, wherein all thymines (t) of saidSEQ ID NO: 16 are substituted by uracils (u), wherein said nucleic acidmolecule is an antisense oligonucleotide, and wherein said antisenseoligonucleotide is a 2′-O-methyl-oligoribonucleotide. Thus, preferably,said nucleic acid molecule comprises or consists of a sequence as setforth in SEQ ID NO: 16, wherein all thymines (t) of said SEQ ID NO: 16are substituted by uracils (u), wherein said nucleic acid molecule is a2′-O-methyl-RNA with full phosphorothioate backbone antisenseoligonucleotides (2′-O-Met). Thus, in an again a further very preferredembodiment of the present invention, said nucleic acid moleculecomprises or consists of a sequence as set forth in SEQ ID NO: 16,wherein all thymines (t) of said SEQ ID NO: 16 are substituted byuracils (u), wherein said nucleic acid molecule is an antisenseoligonucleotide, and wherein said antisense oligonucleotide is a2′-O-methyl-phosphorothioate oligoribonucleotide.

In a further very preferred embodiment of the present invention, saidnucleic acid molecule comprises or consists of a sequence as set forthin SEQ ID NO: 17, wherein all thymines (t) of said SEQ ID NO: 17 aresubstituted by uracils (u), wherein said nucleic acid molecule is anantisense oligonucleotide, and wherein said antisense oligonucleotide isa 2′-O-methyl-oligoribonucleotide. Thus, preferably, said nucleic acidmolecule comprises or consists of a sequence as set forth in SEQ ID NO:17, wherein all thymines (t) of said SEQ ID NO: 17 are substituted byuracils (u), wherein said nucleic acid molecule is a 2′-O-methyl-RNAwith full phosphorothioate backbone antisense oligonucleotides(2′-O-Met). Thus, in a further very preferred embodiment of the presentinvention, said nucleic acid molecule comprises or consists of asequence as set forth in SEQ ID NO: 17, wherein all thymines (t) of saidSEQ ID NO: 17 are substituted by uracils (u), wherein said nucleic acidmolecule is an antisense oligonucleotide, and wherein said antisenseoligonucleotide is a 2′-O-methyl-phosphorothioate oligoribonucleotide.

The terms “morpholino oligomer” or “PMO” (phosphoramidate- orphosphorodiamidate morpholino oligomer) are known to the skilled personin the art and have been described in detail (Singh Y, et al. 2010, ChemSoc Rev 39:2054-2070, as well in U.S. Pat. Nos. 5,698,685, 5,217,866,5,142,047, 5,034,506, 5,166,315, 5,521,063, 5,506,337, 8,076,476,8,299,206 and 7,943,762, all of which are incorporated herein byreference).

Other forms of AONs that may be used to this effect are AON sequencescoupled to small nuclear RNA molecules such as U1 or U7 in combinationwith a viral transfer method based on, but not limited to, lentivirus oradeno-associated virus (Denti, M A, et al., Hum Gene Ther, 200819(6):601-608; Goyenvalle, A, et al., Science 2004 306:1796-1799;WO11113889; WO06021724).

The invention also relates to a composition comprising a nucleic acidmolecule (or AON) of the invention in a pharmaceutically acceptablecarrier. In addition to AONs, pharmaceutical compositions of the presentinvention may also include a pharmaceutically or physiologicallyacceptable carrier such as saline, sodium phosphate, etc. Thecompositions will generally be in the form of a liquid, although thisneed not always be the case. Suitable carriers, excipients and diluentsinclude lactose, dextrose, sucrose, sorbitol, mannitol, starches, gumacacia, calcium phosphates, alginate, tragacanth, gelatin, calciumsilicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose,water syrup, methyl cellulose, methyl and propylhydroxybenzoates,mineral oil, etc. The formulations can also include lubricating agents,wetting agents, emulsifying agents, preservatives, buffering agents,etc. In particular, the present invention involves the administration ofAONs and is thus somewhat akin to gene therapy. Those of skill in theart will recognize that nucleic acids are often delivered in conjunctionwith lipids (e.g. cationic lipids or neutral lipids, or mixtures ofthese), frequently in the form of liposomes or other suitable micro- ornano-structured material (e.g. micelles, lipocomplexes, dendrimers,emulsions, cubic phases, etc.).

The compositions of the invention are generally administered byinjection, e.g. intravenously, subcutaneously or intramuscularly,although other types of administration are not precluded, e.g.inhalation, topical, per os, etc. Injectable preparations, for example,sterile injectable aqueous or oleaginous suspensions may be formulatedaccording to the known art using suitable dispensing or wetting agentsand suspending agents. The sterile injectable preparation can also be asterile injectable solution or suspension in a nontoxic parenterallyacceptable diluent or solvent, for example, as a solution in1,3-butanediol. While delivery may be either local (i.e. in situ,directly into tissue such as muscle tissue) or systemic, usuallydelivery will be local to affected muscle tissue, e.g. to skeletalmuscle, smooth muscle, heart muscle, etc. Depending on the form of theAONs that are administered and the tissue or cell type that is targeted,techniques such as electroporation, sonoporation, a “gene gun”(delivering nucleic acid-coated gold particles), etc. may be employed.

One skilled in the art will recognize that the amount of an AON to beadministered will be an amount that is sufficient to induce ameliorationof unwanted disease symptoms. Such an amount may vary inter aliadepending on such factors as the gender, age, weight, overall physicalcondition, of the patient, etc. and may be determined on a case by casebasis. The amount may also vary according to the other components of atreatment protocol (e.g. administration of other medicaments such assteroids, etc.). Generally, a suitable dose is in the range of fromabout 1 mg/kg to about 100 mg/kg, and more usually from about 2 mg/kg toabout 10 mg/kg. If a viral-based delivery of AONs is chosen, suitabledoses will depend on different factors such as the viral strain that isemployed, the route of delivery (intramuscular, intravenous,intra-arterial or other), but may typically range from 10e10 to 10e12viral particles/kg. Those of skill in the art will recognize that suchparameters are normally worked out during clinical trials. Further,those of skill in the art will recognize that, while disease symptomsmay be completely alleviated by the treatments described herein, thisneed not be the case. Even a partial or intermittent relief of symptomsmay be of great benefit to the recipient. In addition, treatment of thepatient is usually not a single event. Rather, the AONs of the inventionwill likely be administered on multiple occasions, that may be,depending on the results obtained, several days apart, several weeksapart, or several months apart, or even several years apart.

With reference to the treatment of Pompe patients with the c.-32 IVS1-13T>G mutation, the methods of the present invention can be implemented inany of several different ways. For example, the AONs of the presentinvention may be administered together with AONs designed toremove/include other exons from other genes (e.g. in a single mixture,or in separate mixtures but administered in close temporal proximity,such as one directly after the other-in any order-with only a fewminutes or hours between administrations). Alternatively, a patient whois already under treatment using e.g. gene therapy or enzyme replacementtherapy or chaperone therapy may be treated by the methods of theinvention. In other words, the AONs of the invention may be administeredto a patient who is already or has been receiving another treatment, butis still in need of further amelioration of the functional capabilitiesof the GAA molecules produced as a result of the other treatment.

If the AONs of the present invention are to be administered with AONsdesigned for purposes other than to improve inclusion of exon 2 of theGAA mRNA, one possible route of administration is to include sequencesencoding from both types of AONs (those designed to incorporate exon 2of the GAA pre-mRNA and those designed for another reason) in a singlevector that is administered to a patient. Those of skill in the art willrecognize that several vectors are available for use in deliveringnucleic acid sequences so that the nucleic acid sequences may betranscribed in vivo within the recipient. Examples of such vectorsinclude but are not limited to various vectors derived from attenuatedviruses such as retroviral vectors, adenoviral vectors, adeno-associatedviral vectors, HIV and influenza virus vectors, etc. Vectors based onattenuated bacteria might also be employed, e.g. mycobacterial basedvectors. Those of skill in the art will recognize that if these types ofmethods are used, it may be preferable to avoid multiple administrationswhich could result in an adverse immune response to the vector.

The individuals or patients treated by the methods described herein aretypically mammals, usually humans. However, this need not always be thecase. Veterinary applications of this technology are also contemplated.

The following examples serve to further illustrate the invention butshould not be interpreted so as to limit the invention in any way.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an agarose gel showing the lack of exon 2 in GAA mRNAs fromc.-32 IVS1-13 T>G cells. The presence of exon 2 was assessed by nestedRT-PCR (PCR1 from exon 1 to exon 3, followed by PCR2 from exon 2 to exon3) using RNA samples extracted from either primary fibroblasts of apatient with the c.-32 IVS1-13 T>G, (Lane 1) or fibroblasts from ahealthy individual (Lane 2). The amplicon of 670 base pairs testifyinginclusion of exon 2 in the final GAA mRNA was not detected in the RNAextract from c.-32-13T>G cells.

FIG. 2 is a schematic representation of the spliceosome complex.

FIG. 3 is a schematic representation of splicing motives involved in thedefinition of exon 2 of the GAA pre-mRNA and consequences in thepresence of the c.-32 IVS1-13 T>G mutation.

FIG. 4 represents the antisense target sequences for unwinding theacceptor splice site of exon 2 in c.-32 IVS1-13 T>G pre-mRNAs.

FIG. 5A is an agarose gel showing inclusion of exon 2 in GAA mRNAs fromcells with the c.-32-13T>G mutation, after transfection withtricyclo-DNA antisense oligonucleotides in accordance with the presentinvention. The presence of exon 2 was assessed by nested RT-PCR (PCR1from exon 1 to exon 4, followed by PCR2 within exon 2 giving rise to anamplicon of 545 base pairs). Lane 1 is a negative control where the RNAextract is replaced by H₂O. Lane 2 is untreated c.-32-13T>G cells. Lane3 is healthy cells. Lanes 4, 5 and 6 are c.-32-13T>G cells transfectedwith AON1(tc-PS-DNA) having SEQ ID NO: 14 (and annealing to SEQ ID NO:2), AON2(tc-PS-DNA) having SEQ ID NO: 15 (and annealing to SEQ ID NO: 3)and AON3(tc-PS-DNA) having SEQ ID NO: 16 (and annealing to SEQ ID NO:4), respectively.

FIG. 5B is a Western blot analysis of GAA protein expression aftertransfection with tricyclo-DNA antisense oligonucleotides in accordancewith the present invention. Lane 1 is healthy cells. Lane 2 is untreatedc.-32-13T>G cells. Lanes 3, 4 and 5 are c.-32-13T>G cells transfectedwith AON1(tc-PS-DNA) having SEQ ID NO: 14 (and annealing to SEQ ID NO:2), AON2(tc-PS-DNA) having SEQ ID NO: 15 (and annealing to SEQ ID NO: 3)and AON3(tc-PS-DNA) having SEQ ID NO: 16 (and annealing to SEQ ID NO:4), respectively. The acid alpha-glucosidase is revealed as a protein ofabout 70 kDa in lanes 1, 3, 4 and 5.

FIG. 6 is an agarose gel showing inclusion of exon 2 in GAA mRNAs incells with the c.-32-13T>G mutation, after transfection with2′-O-methyl-RNA with full phosphorothioate backbone antisenseoligonucleotides (2′-O-Met) in accordance with the present invention.The presence of exon 2 was assessed by nested RT-PCR (PCR1 from exon 1to exon 3, followed by PCR2 from exon 2 to exon 3 giving rise to anamplicon of 670 base pairs testifying inclusion of exon 2 in the GAAmRNA). Lane 1 is untreated c.-32-13T>G cells. Lanes 2, 3 and 4 arec.32-13T>G cells transfected with AON1(2′-O-Met) having SEQ ID NO: 14(and annealing to SEQ ID NO: 2), AON2(2′-O-Met) having SEQ ID NO: 15(and annealing to SEQ ID NO: 3) and AON3(2′-O-Met) having SEQ ID NO: 16(and annealing to SEQ ID NO: 4), respectively.

FIG. 7 is a schematic representation of the U7opt snRNA harbouring anantisense sequence targeting the exon 2 sequence spanning fromnucleotide 3 to nucleotide 31 (U7opt-AS[+3;+31]). This U7opt snRNA withthe antisense oligonucleotide is also shown in SEQ ID NO:6 (excludingthe SmOPT) and SEQ ID NO: 18 (including the SmOPT as depicted in FIG. 7and named U7opt-AS[+3;+31]).

FIG. 8A is an agarose gel showing inclusion of exon 2 in GAA mRNAs incells with the c.-32-13T>G mutation, after transduction with alentivector (LV) encoding U7opt-AS[+3;+31] of SEQ ID NO: 18). Thepresence of exon 2 was assessed by RT-PCR (from exon 1 to exon 3) givingrise to an amplicon of 635 base pairs testifying inclusion of exon 2 inthe GAA mRNA. Lane 1 is healthy cells. Lane 2 is untreated c.-32-13T>Gcells. Lanes 3, 4 and 5 are c.-32-13T>G cells (25×10³ cells) suppliedwith either 10 μl, 5 μl or 1 μl of LV(U7opt-AS[+3;+31]), respectively.

FIG. 8B is a Western blot analysis of GAA protein expression aftertransduction with LV(U7opt-AS[+3;+31]) in accordance with the presentinvention. Lane 1 is healthy cells. Lane 2 is untreated c.-32-13T>Gcells. Lane 3 is c.-32-13T>G cells (25×10³ cells) supplied with 1 μl ofLV(U7opt-AS[+3;+31]). The acid alpha-glucosidase is revealed as aprotein of about 70 kDa in lanes 1 and 3.

EXAMPLES Example 1

Pompe disease (also called GSD II—Glycogen Storage Disease II) is aninherited recessive disorder caused by mutations in the GAA geneencoding the acid alpha-glucosidase enzyme (also called lysosomalalpha-glucosidase or α-1,4-glucosidase), leading to engorged lysosomalglycogen accumulation and progressive muscular weakness affectingproximal limb and respiratory muscles. The disease encompasses a broadspectrum of clinical phenotypes, which differ in terms of age of onset,extent of organ involvement, and rate of progression: Infantile forms(incidence: 1/140,000) usually correlate with a more aggressive diseasecourse and most patients die by age one; Children and adult forms(incidence: 1/60,000) are more variable across patients; alwaysresulting in loss of ambulation, ventilation support and prematuremortality.

Hundreds of GAA mutations have been identified, but some are more commonamong given ethnic groups. Among them, the c.-32 IVS1-13 T>G mutation isfound in over half of all adult Caucasian patients. It is a splicemutation lessening dramatically the normal inclusion of the second exonof the GAA gene. The foremost outcome of such mis-splicing is thatensuing A2-GAA mRNAs (more than 90% of the GAA transcripts resultingfrom the mutated gene) lack their starting codon and thus cannot betranslated into even semi-functional proteins (FIG. 1).

Splice mutations are many and varied. Most severe (e.g. totallydisabling the splicing reactions) are those that typically affect theconsensus donor (GU) or acceptor (AG) splicing sites at the 5′ and 3′ends of introns, thus impairing transesterification reactions. Mutationswhich affect cis-acting regulatory elements (silencers or enhancers) andother RNA features influencing splicing are usually less severe giventhat resulting inhibition is often incomplete and some normaltranscripts can still be synthesised. The T to G transition provoked bythe c.-32 IVS1-13 mutation is one of those. It is located within thepyrimidine tract (also called polypyrimidine—Py), a series ofpyrimidines (y) among the branch site (in humans the branch consensus isyUnAy) and the AG at the 3′ end of the intron, which allows therecruitment of the dimeric U2 auxiliary factor protein or U2AF,identified as a major site of splicing regulation: the U2AF 65-kDasubunit interacts with the pyrimidine tract, while the smaller 35-kDasubunit directly contacts the 3′ splice site sequence (FIG. 2).Therefore, a common explanation for the effect of the c.-32 IVS1-13mutation might be that the T to G transition weakens the strength of thepolypyrimidine tract, which could not any more efficiently recruit U2AF.Nonetheless, the fact that the Py consensus varies somewhat in lengthand sequence for every intron does not support such an assumption.Instead we hypothesised that the T to G transition might have alteredthe correct removal of intron 1 by modifying the secondary structure ofthe pre-mRNA at the level of its 3′ acceptor splice site, thus making itineffective. In order to investigate this hypothesis and develop aninterventional splice correction therapy for these patients (e.g. havingthe c.-32 IVS1-13 mutation), we analysed the definition of theintron1/exon 2 boundary of the GAA gene and its predicted secondarystructures by using the Human-Splicing-Finder (HSF) (www.umd.be/HSF/)and the RNAfold software of the ViennaRNA Web Services. FIG. 3 shows thelocation of the key determinants of splicing at the intron 1/exon 2boundary, such as the branch point (cugAg), the pyrimidine tract(ccgcuuucuucuccc), the 3′ splice site (SA1). It also shows the putativeexonic splicing enhancer sites (ESE) to which splicing activatorproteins bind, increasing the probability that a nearby site will beused as a splice junction. The HSF analysis revealed an unexpectedfeature of the exon 2: its 5′ region (to be precise the first 40nucleotides) was devoid of ESE sites, but displayed at least three ESSs(exonic splice silencer), which might bind splicing repressors such asthe heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1). The hnRNP A1proteins are able to bind single stranded RNAs in a cooperative way,which can unwind RNA secondary structures and preferentially spreads ina 3′ to 5′ direction to eventually displace SR proteins bound at ESEsites. Interestingly, the RNAfold analysis showed major differences forthe predicted secondary structures of the region spanning fromnucleotide −30 to +39 in either normal sequence or mutated sequence (T>Gin −13). In both cases, MFE (minimum free energy) predicted structuresshowed that this particular region of the pre-mRNA [−30;+39] couldlikely form a hairpin, although such a structure was reinforced in themutant: for the normal sequence, the free energy of the thermodynamicensemble was about −18.38 kcal/mol, the frequency of the MFE structurein the ensemble was 20.46%, and the ensemble diversity was 22.37; forthe mutant sequence, the free energy of the thermodynamic ensemble was−22.02 kcal/mol, the frequency of the MFE structure in the ensemble was36.35%, and the ensemble diversity was 9.68. These predictive structuresallowed us to propose an explanation for the mechanism by which the T>Gmutation takes action on the correct splicing of exon 2. First, innormal, the pre-mRNA around the acceptor splice site tends to form ahairpin potentially hindering the splice reaction. Fortunately, thestrength of this hairpin would be reasonably weak and could be easilyunwound by hnRNP A1 not competing with SR proteins bound a littlefurther at 3′. Second, in mutant, the same region forms a strong hairpinthat cannot be overcome by hnRNP A1. As a result, the high base-pairingprobability of the nucleotides involved in this secondary structuremakes almost inaccessible some crucial consensus sequences at the 3′ endof intron 1, such as the acceptor splice site, the pyrimidine tract forU2AF recruitment and the branch site. Accordingly with theseassumptions, we proposed a method for rescuing full length GAA mRNA inpatient cells with the c.-32 IVS1-13 T>G mutation by using antisenseoligonucleotides (tricyclo-DNA) that were designed to anneal the 5′region of exon 2 (FIG. 4), which is involved in the putative hairpindescribed above, hence allowing to set free the acceptor splice site ofexon 2 and its key upstream cis-elements (Py and Branch site).

Results

Fibroblasts isolated from skin biopsies from GSD II patients with thec.-32 IVS1-13 mutation (the second mutation was c.546 T>G annealing the3′ donor splice site of exon 2) were grown in tissue culture andtransfected with different amounts of tc-PS-DNA oligonucleotides (AON-1;AON-2; AON-3). Two days after transfection, cells were harvested andtotal RNA extracted in order to carry out RT-PCR analysis for assessingthe integrity of the GAA mRNA. FIG. 5 shows subsequent ampliconsobtained by using nested PCR with different sets of primers designed toassess the presence of exon 2 in the final mRNA. As expected, the set ofprimers (Fex1-Rex3 followed by F2-R3) allowed the detection of anamplicon of about 670 base-pairs encompassing exon 2. Such amplificationdid not occurred when using patient cells thus confirming that inclusionof exon 2 in the final mRNA was dramatically affected by the context ofthe mutation. However, in the presence of whatever tested AON (AON-1;AON-2; AON-3) targeting the [+3;+31] region, an amplicon of 670 bp wasdetected straightforward indicative of GAA-mRNA rescue in patient cells(FIG. 5A). Efficient translation of the rescued GAA mRNA was furtherconfirmed by Western blot analysis using total protein extracts intreated cells (FIG. 5B).

These experiments demonstrate that the c.-32 IVS1-13 T>G mutation can berescue by means of splice-switching approaches using tricyclo-DNAoligomers in accordance with the present invention.

Example 2

Experiments similar to those described in Example 1 were carried outusing 2′-O-Met oligomers (FIG. 6) thus confirming that differentchemistries for the synthesis of synthetic antisense oligonucleotidescan be used for GAA rescue in c.-32 IS1-13 GSD II cells.

Example 3

Experiments similar to those described in Examples 1 and 2 were carriedout using the U7 system (FIG. 7) thus confirming that further antisensechemistries such as engineered snRNAs can be used for GAA rescue inc.-32 IS1-13 GSD II cells (FIG. 8).

Material & Methods

Cells:

Cultures of primary fibroblasts were derived from cutaneous biopsies ofGSD II patients (Cell Bank of the Hospital Cochin). Reactives andculture media were products from GIBCO-Invitrogen unless otherwiseindicated.

Skin fibroblasts were dissociated by incubation of cutaneous sampleswith collagenase type 1A (Sigma) and cultured in DMEM medium containing10% foetal bovine serum (Sigma), 100 units/ml of penicillin, 100 μg/mlof streptomycin. When needed, these cells were converted into myogenicprogenitors by transduction with a lentivector encoding for adoxycyclin-inducible MyoD gene. About 600 to 1200 viral particles percell were incubated with fibroblasts for at least 4 hours. Transducedmyo-fibroblasts and fibroblasts were used for transfection studies usingantisense oligonucleotides.

Antisense Sequences Targeting the Exon 2 of the GAA Gene Product:

We designed 3 antisense sequences capable of annealing the 5′ region ofexon 2 of the human acid α-glucosidase pre-mRNA: first from nucleotide+3 to nucleotide +17, second from position +7 to +21 and third fromposition +17 to +31.

Antisense oligonucleotides were synthesised as 2′-O-Met oligomers andtricyclo (tc)-DNAs. Antisense sequences were also introduced into theU7OPT gene system by PCR as previously described (Goyenvalle and al.,2004). The resulting U7OPT-AS cassette was introduced in the pRR1 vector(for lentivector production) at the NheI and XbaI sites of restriction.The synthesis of tricyclo-nucleosides is known in the art, for exampleas described in Steffens R, et al., 1997, Helv Chim Acta 80:2426-2439;Renneberg D, et al., J Am Chem Soc, 2002, 124:5993-6002; WO 2013/053928;and references cited therein). The synthesis of2′-O-methyl-phosphorothioate oligoribonucleotide (2′-O-Met) are alsoknown for the skilled person in the art (WO 2013/112053 and referencescited therein).

Transfection:

We used two concentrations (10 μg and 25 μg) of the three antisenseoligonucleotides (AONs) to transfect fibroblasts from GSDII patients.Cells (20×10³ to 60×10³) were plated into P6 wells. For each well, 10 or25 μg of AON were diluted in 90 μl of DMEM, complexed with 12 μl ofoligofectamin and incubated for 20 minutes. After addition of 800 μl ofthe free medium to the AON-mix, cells are incubated at 37° C. with thismix and diluted four hours later into another 1 ml of the proliferationmedium.

Lentivector Production:

LV(U7opt-AS[+3;+31]) were produced as previously described (Dull T, etal.; J Virol, 1998, 72(11): 8463-8471; Benchaouir R., et al., Cell StemCell, 2007, 1(6):646-657).

Transduction:

Fibroblasts from GSDII patients (25×10³ cells) were incubated in 100 μlof DMEM supplied with different amounts (10; 5 or 1 μl) of aconcentrated preparation of LV(U7opt-AS[+3;+31]) for 4 hours at 37° C.Then, cells were expanded in proliferation medium (DMEM supplementedwith 10% of foetal bovine serum).

Nested PCR:

48 h after transfection, cells were collected and mRNA was extractedusing RNeasy kit according to the manufacturer's instructions (Qiagen).0.5 to 2 ng of total RNA sample was reverse transcribed into cDNA withthe Super-Script II RT (SSIIR Invitrogen) primed by random hexamersaccording to the manufacturer. Exon 2 inclusion in the GAA mRNA wasanalyzed by nested RT-PCR. First PCR amplification was performed using 3μg of cDNA in a 50-μl reaction with the external primers surrounding theexon 2 (Fex1-Rex3). The primary synthesis was performed with 30 cyclesof 94 ° C. (30 seconds), 58 ° C. (1 minute), and 72 ° C. (2 minutes).Three micro litres of the first reaction were then reamplified in nestedPCRs by 25 cycles of 94 ° C. (30 seconds), 58 ° C. (1 minute), and 72 °C. (2 minutes) using the internal primers annealing inside the exon 2.The products of PCR were separated by electrophoresis in a 1.5% agarosegel stained with ethidium bromide.

Fex1: (SEQ ID NO: 7) 5′ AGCTGACGGGGAAACTGAG 3′ Rex4: (SEQ ID NO: 8) 5′AAGGTCCCGGTTCCACAG 3′ Rex3: (SEQ ID NO: 9) 5′ GCTCCTCGGAGAACTCCAC 3′Fex2: (SEQ ID NO: 10) 5′ CTGTCCAGGCCATCTCCA 3′ Rex2: (SEQ ID NO: 11) 5′AGTCTCCATCATCACGTCCA 3′

Western Blot Analysis:

The molecular mass of the human acid alpha-glucosidase was analyzed atday 3 post-transfection in the healthy, deficient and Tc-DNA treatedfibroblasts. The cells were washed twice with PBS, trypsinized andpelleted. Protein lysates from cells were resolved by SDS-PAGE andtransferred overnight to nitrocellulose. The specific protein wasdetected using a goat polyclonal antibody raised against human GAA. Theimmune complexes were visualized using a rabbit anti-goat immunoglobulinantiserum and chemiluminescence.

Histochemical Analysis:

Glycogen storage was determined by PAS staining. Control and treatedcultured cells were rinsed in PBS for 5 min followed by incubation inSchiff reagent for 15 min at room temperature. They were analyzed bylight microscopy.

1. A nucleic acid molecule 10 to 50 nucleotides in length, complementaryto a nucleotide sequence of the acid alpha-glucosidase pre-mRNA, saidnucleic acid being able to correct the splicing of exon 2 of the acidalpha-glucosidase pre-mRNA.
 2. The nucleic acid molecule according toclaim 1, wherein said nucleic acid molecule is able to correct thesplicing of exon 2 of the human acid alpha-glucosidase pre-mRNAdisplaying the c.-32-13T>G mutation.
 3. The nucleic acid moleculeaccording to claim 1, comprising a nucleotide sequence complementary toa sequence comprised in the region defined by positions [+3;+45] of exon2 of the GAA gene.
 4. The nucleic acid molecule according to claim 1,wherein said nucleotide sequence is complementary to one or more of thesequences defined by positions 3-17, 7-21 or 17-31 in SEQ ID NO:1 or ina sequence having at least 90% homology with SEQ ID NO:1.
 5. The nucleicacid molecule according to claim 1, wherein said nucleotide sequence iscomplementary to one of the sequences comprising SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:12 and SEQ ID NO:13.
 6. Thenucleic acid molecule according to claim 1, wherein said nucleic acidmolecule comprises a nucleotide sequence complementary to a sequencecomprised in the region defined by positions [+3;+31] of exon 2 of thehuman GAA gene (SEQ ID NO:1).
 7. The nucleic acid molecule according toclaim 1, wherein said nucleic acid molecule comprises or is an antisenseoligonucleotide.
 8. The nucleic acid molecule according to claim 1,wherein said nucleic acid molecule comprises oligodeoxyribonucleotides,oligoribonucleotides, morpholinos, tricyclo-DNA-antisenseoligonucleotides, tricyclo-phosphorothioate DNA-antisenseoligonucleotides, LNA oligonucleotides and U7- or U1-modified nucleicacid molecule.
 9. The nucleic acid molecule according to claim 1,wherein said nucleic acid molecule comprises tricyclo-DNA-antisenseoligonucleotides.
 10. The nucleic acid molecule according to claim 1,wherein said nucleic acid molecule comprises tricyclo-phosphorothioateDNA-antisense oligonucleotides.
 11. The nucleic acid molecule accordingto claim 1, wherein said nucleic acid molecule comprises a sequenceselected from SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ IDNO:
 17. 12. The nucleic acid molecule according to claim 1, wherein saidnucleic acid molecule is linked to a modified U7 snRNA.
 13. A method forthe treatment of Pompe disease, comprising administering the nucleicacid molecule of claim 1 to a patient in need thereof.
 14. The methodaccording to claim 13, wherein the patient with Pompe disease harborsthe c.-32 IVS1-13 T>G or an IVS1.-3 C>N mutation in the GAA gene.
 15. Apharmaceutical composition comprising a nucleic acid molecule accordingto claim 1, in a pharmaceutically acceptable carrier.
 16. The nucleicacid molecule according to claim 6, wherein said sequence comprised inthe region defined by positions [+3;+31] of exon 2 of the human GAA gene(SEQ ID NO:1) has a length from at least 10 to at most 29 nucleotides.17. The method according to claim 13, wherein the patient with Pompedisease harbors the c.-32-13 T>G mutation in the human GAA gene.
 18. Thenucleic acid molecule according to claim 1, wherein all tc-nucleosidesof said tricyclo-phosphorothioate DNA-antisense oligonucleotides arejoined by inter nucleoside phosphorothioate linkages.
 19. The nucleicacid molecule according to claim 1, wherein said nucleic acid moleculecomprises or consists of 2′-O-methyl-RNAs.
 20. The nucleic acid moleculeaccording to claim 1, wherein said nucleic acid is a 2′-O-methyl-RNAwith full phosphorothioate backbone.